Process for purifying semaglutide and liraglutide

ABSTRACT

The present invention provides improved processes for purifying semaglutide or liraglutide. Semaglutide or liraglutide is purified via two sequential RP-HPLC purifications followed by a salt-exchange step, where a pH is kept constant in the first and second purification steps. In particular, the processes utilize a halogenated solvent in a sample preparation step, which provides better solubility and an environment suitable for decarboxylation for crude semaglutide or liraglutide prior to a RP-HPLC purification.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/829,725 filed Mar. 25, 2020, which claims priority to U.S.Provisional Application No. 62/823,147 filed Mar. 25, 2019, each ofwhich is incorporated herein in its entirety for all purpose.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is Sequence_Listing_for_050760_54002US_ST25.txt.The text file is 1.46 KB, was created on Feb. 2, 2021, and is beingsubmitted electronically via EFSWeb.

BACKGROUND OF THE INVENTION

Human glucagon-like peptide-1 (GLP-1) receptor agonists includeexenatide, liraglutide, dulaglutide, lixisenatide, and semaglutide.Among them, semaglutide (trade name Ozempic) and liraglutide (trade nameVictoza), analogs of human glucagon-like peptide-1 (GLP-1), areindicated as an adjunct to diet and exercise to improve glycemic controlin adults with type 2 diabetes mellitus.

Semaglutide includes a peptide having SEQ ID NO: 1, which has beenengineered to be 94% homologous to native human GLP-1 by substitutingalpha-aminoisobutyric acid (Aib) for alanine at position 8 and argininefor lysine at position 34. Semaglutide is further derived from the notedengineered peptide by attaching a C-18 fatty diacid through a PEGmodified glutamic acid hydrophilic spacer at the lysine residue ofposition 26. Accordingly, the structure of semaglutide with notedmodifications based on native human GLP-1 is shown below:

Semaglutide has a molecular formula of C₁₈₇H₂₉₁N₄₅O₅₉ and a molecularweight of 4113.58 Daltons. It was originally developed by Novo Nordisk,and approved by the U. S. Food and Drug Administration (FDA) in December2017 under the trade name “Ozempic”.

Liraglutide includes a peptide having SEQ ID NO: 2, which has beenengineered to be 97% homologous to native human GLP-1 by substitutingarginine for lysine at position 34. Liraglutide is further derived fromthe noted engineered peptide by attaching a C-16 fatty acid (palmiticacid) through a glutamic acid spacer at the lysine residue of position26. Accordingly, the structure of liraglutide with noted modificationsbased on native human GLP-1 is shown below:

Liraglutide has a molecular formula of C₁₇₂H₂₆₅N₄₃O₅₁ and a molecularweight of 3751.2 Daltons. It was originally developed by Novo Nordisk,and approved by the U. S. Food and Drug Administration (FDA) in January2010 under the trade name “Victoza”.

U.S. Pat. No. 9,422,330 B2 discloses methods for purifying crudesemaglutide produced synthetically and crude liraglutide produced fromyeast fermentation, respectively. As described in Example 4 of U.S. Pat.No. 9,422,330, the crude semaglutide was diluted with water andsubjected to a RP-HPLC purification, where stationary phase wasoctadecyl-dimethylsilyl silica, which was initially equilibrated with amobile phase of 10 mmol/kg citrate buffer/125 mmol/kg NaCl/25% ethanolat pH 7.4. The column was first washed by a linear gradient of 25-48%ethanol (10 mmol/kg citrate buffer, 125 mmol/kg NaCl, pH 4.5), thenisocratic washed at EtOH 48% and pH 4.5. The target semaglutide was theneluted by a linear pH gradient elution from pH 4.5 to pH 7.4 with amobile phase including 10 mmol/kg citrate buffer, 125 mmol/kg NaCl andEtOH 48%. The chromatographic temperature for purifying semaglutide waskept at 50° C. As described in Example 6 of U.S. Pat. No. 9,422,330, thecrude liraglutide was diluted with water and subjected to a RP-HPLCpurification, where stationary phase was octadecyl-dimethylsilyl silica,which was initially equilibrated with a mobile phase of 20 mM Trisbuffer/20% ethanol at pH 7.5. The column was first washed with theequilibration solution and eluted with a linear gradient followed by anisocratic gradient with a mobile phase including ethanol and 20 mMcitrate buffer at pH 5.1. The target liraglutide was then eluted by alinear pH gradient elution from pH 5.1 to pH 4.0 with a mobile phaseincluding ethanol and 20 mM citrate buffer. The chromatographictemperature for purifying liraglutide was kept at 60° C.

U.S. Patent Publication No. 2015/0051372 A1 discloses a three-stepmethod for purifying solid-phase synthetic crude liraglutide: sampletreatment followed by three RP-HPLC purifications. In the sampletreatment, crude liraglutide was dissolved in a mixture of acetonitrileand water (e.g., 20% acetonitrile/80% water by volume). The preparedsample was subjected to a first RP-HPLC purification, where mobile phaseA (MPA) was 0.1% TFA/isopropanol/water; mobile phase B (MPB) was 0.1%TFA/acetonitrile; stationary phase was octylsilane bonded silica; andthe gradient was a linear gradient. The fraction obtained by the firstpurification was then subjected to a second RP-HPLC purification, whereMPA was 0.05-0.15% perchloric acid in water; MPB was 0.05-0.15%perchloric acid in acetonitrile; stationary phase was cyanosilane bondedsilica; and the gradient was a linear gradient. The purified liraglutidewas finally subjected to a third RP-HPLC purification fordesalinization, where MPA was 0.05% aqueous ammonia solution; MPB wasacetonitrile, stationary phase was octylsilane bonded silica; and thegradient was a linear gradient.

Despite the above described purifying processes, there remains a needfor the development of more efficient and improved processes forpurifying semaglutide or liraglutide. The present disclosure addressesthis need and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a process forpurifying semaglutide, the process including:

-   -   (a) dissolving crude semaglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting purified semaglutide in fractions.

In a second aspect, the present application provides a process forpurifying semaglutide, the process including:

-   -   (a) dissolving crude semaglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting the purified semaglutide fractions, wherein the        RP-HPLC purification system includes a first RP-HPLC        purification and a second RP-HPLC purification; and    -   (c) subjecting the fractions in step (b) to a third RP-HPLC        purification using a mobile phase E including a sodium salt,        water, and acetonitrile; and collecting fractions to obtain        salt-exchanged semaglutide.

In a third aspect, the present invention provides a process forpurifying liraglutide, the process including:

-   -   (a) dissolving crude liraglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting purified liraglutide in fractions.

In a fourth aspect, the present application provides a process forpurifying liraglutide, the process including:

-   -   (a) dissolving crude liraglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting the purified liraglutide fractions, wherein the        RP-HPLC purification system includes a first RP-HPLC        purification and a second RP-HPLC purification; and    -   (c) subjecting the fractions in step (b) to a third RP-HPLC        purification using a mobile phase E including a sodium salt,        water, and acetonitrile; and collecting fractions to obtain        salt-exchanged liraglutide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows elution chromatogram (full view) of the first purificationaccording to Example 1.

FIG. 2 shows elution chromatogram (zoom in) of FIG. 1. The 1^(st) mainfraction was used for the subsequent second purification.

FIG. 3 shows elution chromatogram (full view) of the second purificationaccording to Example 1.

FIG. 4 shows elution chromatogram (zoom in) of FIG. 3. The 2^(nd) mainfraction comprised the purified semaglutide.

FIG. 5 shows elution chromatogram (full view) of the first purificationaccording to Example 2.

FIG. 6 shows elution chromatogram (zoom in) of FIG. 5. The 1^(st) mainfraction was used for the subsequent second purification.

FIG. 7 shows elution chromatogram (full view) of the second purificationaccording to Example 2.

FIG. 8 shows elution chromatogram (zoom in) of FIG. 7. The 2^(nd) mainfraction comprised the purified semaglutide.

FIG. 9 shows elution chromatogram (full view) of the first purificationaccording to Example 3.

FIG. 10 shows elution chromatogram (zoom in) of FIG. 9. The fractions offrom 5 to 13 as indicated were combined as 1P Main Fraction for thesubsequent second purification.

FIG. 11 shows elution chromatogram (full view) of the first purificationaccording to Example 4 or 5.

FIG. 12 shows elution chromatogram (zoom in) of FIG. 11. The fractionsof from 10-15 as indicated were combined as 1P Main Fraction for thesubsequent second purification.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides improved processes for purifyingsemaglutide or liraglutide. Semaglutide or liraglutide is purified viatwo sequential RP-HPLC purifications followed by a salt-exchange step,where a pH is kept constant in the first and second purification steps.In particular, the processes utilize a halogenated solvent in a samplepreparation step, which provides better solubility and an environmentsuitable for decarboxylation for crude semaglutide or liraglutide priorto a RP-HPLC purification. Tables A and B summarize advantages orcharacteristics of the present invention as compared to the processesdisclosed in the art.

TABLES A The Present Invention vs US2015/0051372 ParametersUS2015/0051372 Present Invention Claimed Advantages Solvent System Amixture of acetonitrile A solution including a 1. Provide bettersolubility for the Sample and water halogenated solvent anddecarboxylation Preparation (e.g., TFE, HFIP, TFA) condition for crudesemaglutide or liraglutide; and 2. Prevent retention disturbing duringthe RP-HPLC purification Stationary Two different stationary Only onestationary Simple and convenient Phase phase materials in three phasematerial operations RP-HPLC purifications: throughout three First andthird: octylsilane RP-HPLC purifications: second: cyanosilaneoctylsilane TFE: trifluoroethanol; HFIP: hexafluoroisopropanol; and TFA:trifluroacetic acid.

TABLES B The Present Invention vs U.S. Pat. No. 9,422,330 ParametersU.S. Pat. No. 9,422,330 Present Invention Claimed Advantages Gradient,Semaglutide or liraglutide The pH is kept constant 1. Elution ofsemaglutide pH is eluted with a pH within each purification orliraglutide beyond its gradient, which is close to step (absence of a pHcorresponding pI value its corresponding pI value gradient) prevents thepeptide from precipitation; and 2. Simple and convenient operations

II. Definitions

SEQ ID NO: 1 refers to the amino acid sequence of the engineered peptidein semaglutide. The sequence is represented by:H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

SEQ ID NO: 2 refers to the amino acid sequence of the engineered peptidein liraglutide. The sequence is represented by:H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

“About” means a range of values including the specified value, which aperson of ordinary skill in the art would consider reasonably similar tothe specified value. In some embodiments, the term “about” means withina standard deviation using measurements generally acceptable in the art.In some embodiments, about means a range extending to +/−10% of thespecified value. In some embodiments, about means the specified value.

“RP-HPLC” refers to a reversed phase-high performance/pressure liquidchromatograph.

“Ammonium salt” refers to a class of chemical compounds which arecomposed of a cation of NR₄ ⁺ and an anion, where R groups can behydrogen or alkyl. In one embodiment, an ammonium salt refers to a classof chemical compounds which are composed of a cation of NH₄ ⁺ and ananion. Ammonium salts useful in the present invention include ammoniumformate and ammonium acetate.

“Phosphate salt” refers to a class of chemical compounds which arecomposed of a cation and a phosphate anion, such as the phosphate anion(PO₄ ³⁻), the hydrogenphosphate anion (HPO₄ ²⁻), and thedihydrogenphosphate anion (H₂PO₄ ⁻). A phosphate salt having the anionof PO₄ ³⁻ also refers to a phosphate tribasic salt; a phosphate salthaving the anion of HPO₄ ²⁻ also refers to a phosphate dibasic salt; andphosphate salt having the anion of H₂PO₄ ⁻ also refers to a phosphatemonobasic salt. Phosphate salts useful in the present invention includesodium phosphate tribasic (Na₃PO₄), sodium phosphate dibasic (Na₂HPO₄),sodium phosphate monobasic (NaH₂PO₄), potassium phosphate tribasic(K₃PO₄), potassium phosphate dibasic (K₂HPO₄), potassium phosphatemonobasic (KH₂PO₄), ammonium phosphate tribasic ((NH₄)₃PO₄), ammoniumphosphate dibasic ((NH₄)₂HPO₄), ammonium phosphate monobasic((NH₄)H₂PO₄).

III. Processes A. Purification of Semaglutide

In a first aspect, the present invention provides a process forpurifying semaglutide. The process includes:

-   -   (a) dissolving crude semaglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting purified semaglutide in fractions.

In some embodiments, the halogenated solvent in step (a) is selectedfrom the group consisting of fluorinated acid, fluorinated alcohol,chlorinated solvent, and mixtures thereof.

In some embodiments, the fluorinated alcohol is selected from the groupconsisting of 2,2,2-trifluoroethanol (TFE),1,1,1,3,3,3-hexafluoro-2-propanol (HFIP),2,2,3,3-tetrafluoro-1-propanol, 2,2,3,3,3-pentafluoro-1-propanol,pentafluorophenol, nonafluoro-tert-butyl alcohol,3,3,4,4,4-pentafluoro-1-butanol, 4,4,5,5,5-pentafluoro-1-pentanol,2,2,3,4,4,4-hexafluoro-1-butanol,1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol,1,1,1,3,3,3-hexafluoro-2-phenyl-2-propanol, and mixtures thereof.

In some embodiments, the chlorinated solvent is 2,2,2-trichloroethanol.

In some embodiments, the fluorinated acid is trifluoroacetic acid.

In some embodiments, the solution in step (a) includes 5-35% fluorinatedalcohol and 0.1-1% fluorinated acid by volume. In some embodiments, thesolution in step (a) includes 15-20% 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) and 0.1-1% fluorinated acid by volume. In some embodiments, thesolution in step (a) includes 20-30% 2,2,2-trifluoroethanol (TFE) and0.1-1% fluorinated acid by volume. In some embodiments, the solution instep (a) includes 15-20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and0.1-1% trifluoroacetic acid by volume. In some embodiments, the solutionin step (a) includes 20-30% 2,2,2-trifluoroethanol (TFE) and 0.1-1%trifluoroacetic acid by volume. In some embodiments, the solution instep (a) includes 15-20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and0.1-0.3% trifluoroacetic acid by volume. In some embodiments, thesolution in step (a) includes 20-30% 2,2,2-trifluoroethanol (TFE) and0.1-0.3% trifluoroacetic acid by volume. In some embodiments, thesolution in step (a) includes about 17%1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and about 0.2% trifluoroaceticacid by volume. In some embodiments, the solution in step (a) includesabout 25% 2,2,2-trifluoroethanol (TFE) and about 0.2% trifluoroaceticacid by volume.

In some embodiments, the RP-HPLC purification system of step (b)includes a first RP-HPLC purification and a second RP-HPLC purification;the first RP-HPLC purification is conducted at a pH value of <7; and thesecond RP-HPLC purification is conducted at a pH value of >7.

In some embodiments, the first RP-HPLC purification is conducted at a pHvalue of <7; and the second RP-HPLC purification is conducted at a pHvalue of >about 7. In some embodiments, the first RP-HPLC purificationis conducted at a pH value of <3; and the second RP-HPLC purification isconducted at a pH value of >about 7. In some embodiments, the firstRP-HPLC purification is conducted at a pH value of <3; and the secondRP-HPLC purification is conducted at a pH value of >about 7.5.

In some embodiments, the first RP-HPLC purification uses a mobile phaseA including trifluoroacetic acid and water and a mobile phase Bincluding trifluoroacetic acid and acetonitrile.

In some embodiments, the mobile phase A and mobile phase B include each0.1-1% trifluoroacetic acid by volume. In some embodiments, the mobilephase A and mobile phase B include each 0.1-0.5% trifluoroacetic acid byvolume. In some embodiments, the mobile phase A and mobile phase Binclude each 0.1-0.3% trifluoroacetic acid by volume. In someembodiments, the mobile phase A and mobile phase B include each about0.2% trifluoroacetic acid by volume. In some embodiments, the mobilephase A is a solution of 0.2% trifluoroacetic acid in water; and mobilephase B is a solution of about 0.2% trifluoroacetic acid inacetonitrile.

In some embodiments, the mobile phase B is eluted at a linear gradientof from 5-25% to 80-100% by volume. In some embodiments, the mobilephase B is eluted at a linear gradient of from 5-15% to 90-100% byvolume. In some embodiments, the mobile phase B is eluted at a lineargradient of from 5-25% to 80-100% by volume, wherein the mobile phase Ais a solution of 0.2% trifluoroacetic acid in water; and mobile phase Bis a solution of about 0.2% trifluoroacetic acid in acetonitrile. Insome embodiments, the mobile phase B is eluted at a linear gradient offrom 5-15% to 90-100% by volume, wherein the mobile phase A is asolution of 0.2% trifluoroacetic acid in water; and mobile phase B is asolution of about 0.2% trifluoroacetic acid in acetonitrile. In someembodiments, the mobile phase B is eluted at a linear gradient of fromabout 10% to 95% by volume, wherein the mobile phase A is a solution of0.2% trifluoroacetic acid in water; and mobile phase B is a solution ofabout 0.2% trifluoroacetic acid in acetonitrile. In some embodiments,the mobile phase B is eluted at a linear gradient of from about 10% to95% by volume according to the gradient of Table 1, wherein the mobilephase A is a solution of 0.2% trifluoroacetic acid in water; and mobilephase B is a solution of about 0.2% trifluoroacetic acid inacetonitrile.

In some embodiments, the second RP-HPLC purification uses a mobile phaseC including an ammonium salt or a phosphate salt and a mobile phase Dincluding acetonitrile. In some embodiments, the second RP-HPLCpurification uses a mobile phase C including an ammonium salt and amobile phase D including acetonitrile. In some embodiments, the secondRP-HPLC purification uses a mobile phase C including a phosphate saltand a mobile phase D including acetonitrile. In some embodiments, thesecond RP-HPLC purification uses a mobile phase C and a mobile phase D,each of which includes an ammonium salt or a phosphate salt. In someembodiments, the second RP-HPLC purification uses a mobile phase C and amobile phase D, each of which includes an ammonium salt. In someembodiments, the second RP-HPLC purification uses a mobile phase Cincluding an aqueous solution of an ammonium salt and a mobile phase Dincluding an aqueous solution of an ammonium salt and acetonitrile. Insome embodiments, the second RP-HPLC purification uses a mobile phase Cincluding an aqueous solution of an ammonium salt and a mobile phase Dbeing acetonitrile.

In some embodiments, the mobile phase C further includes acetonitrile inan amount of from 1% to 10% by volume. In some embodiments, the mobilephase C further includes acetonitrile in an amount of from 3% to 7% byvolume. In some embodiments, the mobile phase C further includesacetonitrile in an amount of about 5% by volume.

In some embodiments, the mobile phase D further includes an aqueoussolution of an ammonium salt in an amount of from 10% to 60% by volume.In some embodiments, the mobile phase D further includes an aqueoussolution of an ammonium salt in an amount of from 20% to 60%, 30% to60%, 40% to 60%, or about 50% by volume. In some embodiments, the mobilephase D further includes an aqueous solution of an ammonium salt in anamount of about 50% by volume.

In some embodiments, the ammonium salt is selected from the groupconsisting of ammonium formate, ammonium acetate, and mixtures thereof.In some embodiments, the ammonium salt is ammonium formate.

In some embodiments, the phosphate salt is selected from the groupconsisting of sodium phosphate tribasic, sodium phosphate dibasic,sodium phosphate monobasic, potassium phosphate tribasic, potassiumphosphate dibasic, potassium phosphate monobasic, ammonium phosphatetribasic, ammonium phosphate dibasic, ammonium phosphate monobasic, andmixtures thereof. In some embodiments, the phosphate salt is selectedfrom the group consisting of sodium phosphate dibasic, sodium phosphatemonobasic, potassium phosphate dibasic, potassium phosphate monobasic,ammonium phosphate dibasic, ammonium phosphate monobasic, and mixturesthereof. In some embodiments, the phosphate salt is selected from thegroup consisting of potassium phosphate dibasic, potassium phosphatemonobasic, and mixtures thereof. In some embodiments, the phosphate saltis a mixture of potassium phosphate dibasic and potassium phosphatemonobasic.

In some embodiments, the mobile phase C includes the ammonium salt orphosphate salt in a concentration of from 5 to 50 mM in water. In someembodiments, the mobile phase C includes the ammonium salt or phosphatesalt in a concentration of from 10 to 40 mM in water. In someembodiments, the mobile phase C includes the ammonium salt or phosphatesalt in a concentration of from 10 to 30 mM, from 15 to 25 mM, or about15 mM in water. In some embodiments, the mobile phase C includes theammonium salt or phosphate salt in a concentration of about 15 mM inwater. In some embodiments, the mobile phase C includes the ammoniumsalt in a concentration of about 15 mM in water. In some embodiments,the mobile phase C includes an aqueous solution of 15 mM ammonium salt.In some embodiments, the mobile phase C includes the phosphate salt in aconcentration of about 20 mM in water. In some embodiments, the mobilephase C includes an aqueous solution of 20 mM phosphate salt.

In some embodiments, the mobile phase C has a pH value of from about 7.5to about 9.0 or from about 7.5 to about 8.5. In some embodiments, themobile phase C has a pH value of from about 7.5 to about 8.5. In someembodiments, the mobile phase C has a pH value of about 8.3.

In some embodiments, mobile phase C includes an aqueous solution ofammonium formate. In some embodiments, mobile phase C includes anaqueous solution of 15 mM ammonium formate. In some embodiments, mobilephase C includes an aqueous solution of 15 mM ammonium formate; and hasa pH value of about 8.3. In some embodiments, mobile phase C includesacetonitrile and an aqueous solution of 15 mM ammonium formate; and hasa pH value of about 8.3, wherein the aqueous solution and acetonitrilehas a ratio of 95 to 5 by volume.

In some embodiments, mobile phase C includes an aqueous solution ofpotassium phosphate dibasic and potassium phosphate monobasic. In someembodiments, mobile phase C includes an aqueous solution of potassiumphosphate dibasic and potassium phosphate monobasic in a concentrationof 20 mM. In some embodiments, mobile phase C includes an aqueoussolution of potassium phosphate dibasic and potassium phosphatemonobasic in a concentration of 20 mM; and has a pH value of about 7.9.In some embodiments, mobile phase C includes acetonitrile and an aqueoussolution of potassium phosphate dibasic and potassium phosphatemonobasic in a concentration of 20 mM; and has a pH value of about 7.9,wherein potassium phosphate dibasic and potassium phosphate monobasichas a ratio of 12 to 1 by weight, and the aqueous solution andacetonitrile has a ratio of 95 to 5 by volume.

In some embodiments, the mobile phase D has a pH value of from about 7.5to about 9.0 or from about 7.5 to about 8.5. In some embodiments, themobile phase D has a pH value of from about 7.5 to about 8.5. In someembodiments, the mobile phase D has a pH value of about 8.5.

In some embodiments, mobile phase D further includes an aqueous solutionof ammonium formate. In some embodiments, mobile phase D furtherincludes an aqueous solution of 15 mM ammonium formate. In someembodiments, mobile phase D further includes an aqueous solution of 15mM ammonium formate; and has a pH value of about 8.3. In someembodiments, mobile phase D includes an aqueous solution of 15 mMammonium formate and acetonitrile; and has a pH value of about 8.3,wherein the aqueous solution and acetonitrile has a ratio of 50 to 50 byvolume.

In some embodiments, mobile phase D is acetonitrile.

In some embodiments, mobile phase C includes an aqueous solution of 15mM ammonium formate and acetonitrile and has a pH value of about 8.3;and mobile phase D includes an aqueous solution of 15 mM ammoniumformate and acetonitrile and has a pH value of about 8.3, wherein theaqueous solution and acetonitrile in the mobile phase C has a ratio of95 to 5 by volume and the aqueous solution and acetonitrile in themobile phase D has a ratio of 50 to 50 by volume.

In some embodiments, mobile phase C includes acetonitrile and an aqueoussolution of potassium phosphate dibasic and potassium phosphatemonobasic in a concentration of 20 mM and has a pH value of about 7.9;and mobile phase D is acetonitrile, wherein potassium phosphate dibasicand potassium phosphate monobasic has a ratio of 12 to 1 by weight, andthe aqueous solution and acetonitrile in the mobile phase C has a ratioof 95 to 5 by volume.

In some embodiments, the mobile phase D is eluted at linear gradientfrom 0-10% to 50-100% by volume. In some embodiments, the mobile phase Dis eluted at linear gradient from 0% to 100% by volume, wherein mobilephase C includes an aqueous solution of 15 mM ammonium formate andacetonitrile and has a pH value of about 8.3; and mobile phase Dincludes an aqueous solution of 15 mM ammonium formate and acetonitrileand has a pH value of about 8.3, wherein the aqueous solution andacetonitrile in the mobile phase C has a ratio of 95 to 5 by volume andthe aqueous solution and acetonitrile in the mobile phase D has a ratioof 50 to 50 by volume. In some embodiments, the mobile phase D is elutedat linear gradient from 0% to 100% by volume wherein mobile phase Cincludes an aqueous solution of 15 mM ammonium formate and acetonitrileand has a pH value of about 8.3; and mobile phase D includes an aqueoussolution of 15 mM ammonium formate and acetonitrile and has a pH valueof about 8.3, wherein the aqueous solution and acetonitrile in themobile phase C has a ratio of 95 to 5 by volume and the aqueous solutionand acetonitrile in the mobile phase D has a ratio of 50 to 50 byvolume. In some embodiments, the mobile phase D is eluted at lineargradient from 5% to 70% by volume, wherein mobile phase C includesacetonitrile and an aqueous solution of potassium phosphate dibasic andpotassium phosphate monobasic in a concentration of 20 mM and has a pHvalue of about 7.9; and mobile phase D is acetonitrile, whereinpotassium phosphate dibasic and potassium phosphate monobasic has aratio of 12 to 1 by weight, and the aqueous solution and acetonitrile inthe mobile phase C has a ratio of 95 to 5 by volume. In someembodiments, the mobile phase D is eluted at linear gradient from 5% to70% by volume, wherein mobile phase C includes acetonitrile and anaqueous solution of potassium phosphate dibasic and potassium phosphatemonobasic in a concentration of 20 mM and has a pH value of about 7.9;and mobile phase D is acetonitrile, wherein potassium phosphate dibasicand potassium phosphate monobasic has a ratio of 12 to 1 by weight, andthe aqueous solution and acetonitrile in the mobile phase C has a ratioof 95 to 5 by volume. In some embodiments, the mobile phase D is elutedat linear gradient from 5% to 70% by volume according to the gradientTable 2, wherein mobile phase C is an aqueous solution of 15 mM ammoniumformate and has a pH value of about 8.3; and mobile phase D isacetonitrile.

In some embodiments, the purified semaglutide in fractions has a pHvalue of from about 7.5 to about 8.5. In some embodiments, the purifiedsemaglutide in fractions has a pH value of from about 8.0 to about 8.5.

The flow rate of the first and second purification depends on the sizeof the RP-HPLC preparation column. In some embodiments, the flow rate ofthe first purification is from about 50 to 200 mL/min, when the columnhas a diameter and length of from 5 cm×22 cm to 8 cm×12.6 cm. In someembodiments, the flow rate of the first purification is from about 100to 160 mL/min, when the column has a diameter and length of from 5 cm×22cm to 8 cm×12.6 cm. In some embodiments, the flow rate of the firstpurification is about 104 mL/min, when the column has a diameter andlength of 5 cm×22 cm. In some embodiments, the flow rate of the firstpurification is about 152 mL/min, when the column has a diameter andlength of 8 cm×12.6 cm. In some embodiments, the flow rate of the firstpurification is from 0.5 to 2.0 mL/min, when the column has a diameterand length of 4.6 mm×250 mm. In some embodiments, the flow rate of thefirst purification is about 1.0 mL/min, when the column has a diameterand length of 4.6 mm×250 mm. In some embodiments, the flow rate of thesecond purification is from about 50 to 200 mL/min, when the column hasa diameter and length of from 5 cm×22 cm to 8 cm×12.6 cm. In someembodiments, the flow rate of the second purification is from about 100to 160 mL/min, when the column has a diameter and length of from 5 cm×22cm to 8 cm×12.6 cm. In some embodiments, the flow rate of the secondpurification is about 152 mL/min, when the column has a diameter andlength of 8 cm×12.6 cm. In some embodiments, the flow rate of the secondpurification is from about 0.3 to 0.5 mL/min, when the column has adiameter and length of 4.6 mm×100 mm. In some embodiments, the flow rateof the second purification is about 0.4 mL/min, when the column has adiameter and length of 4.6 mm×100 mm.

In some embodiments, the crude semaglutide of step (a) is obtained fromsolid-phase synthesis.

In some embodiments, further comprising:

-   -   (c) subjecting the fractions in step (b) to a third RP-HPLC        purification using a mobile phase E comprising a sodium salt,        water, and acetonitrile; and collecting fractions to obtain        salt-exchanged semaglutide.

In some embodiments, the sodium salt is sodium phosphate dibasic.

In some embodiments, the mobile phase E includes an aqueous solution ofsodium phosphate dibasic. In some embodiments, mobile phase E includesan aqueous solution of sodium phosphate dibasic in a concentration of 20mM. In some embodiments, mobile phase E includes an aqueous solution ofsodium phosphate dibasic in a concentration of 20 mM; and has a pH valueof about 8.0. In some embodiments, mobile phase E (as E1) includesacetonitrile and an aqueous solution of sodium phosphate dibasic in aconcentration of 20 mM; and has a pH value of about 8.0, wherein theaqueous solution and acetonitrile has a ratio of 95 to 5 by volume.

In some embodiments, mobile phase E (as E2) includes acetonitrile and anaqueous solution of sodium phosphate dibasic in a concentration of 20mM; and has a pH value of about 8.0, wherein the aqueous solution andacetonitrile has a ratio of 50 to 50 by volume.

In some embodiments, step (c) is conducted by applying the mobile phaseE1 followed by the mobile phase E2, thereby providing a salt-exchangedsemaglutide in fractions.

The flow rate of the third purification depends on the size of theRP-HPLC preparation column. In some embodiments, the flow rate of thethird purification is from 4 to 8 mL/min, when the column has a diameterand length of 10 mm×250 mm. In some embodiments, the flow rate of thefirst purification is about 4.7 mL/min, when the column has a diameterand length of 10 mm×25 cm.

In some embodiments, the salt-exchanged semaglutide in fractions has apH value of from about 7.5 to about 8.0.

In some embodiments, the first, second, and third RP-HPLC purificationshave the same stationary phase.

In a second aspect, the present application provides a process forpurifying semaglutide. The process includes:

-   -   (a) dissolving crude semaglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting the purified semaglutide fractions, wherein the        RP-HPLC purification system includes a first RP-HPLC        purification and a second RP-HPLC purification; and    -   (c) subjecting the fractions in step (b) to a third RP-HPLC        purification using a mobile phase E including a sodium salt,        water, and acetonitrile; and collecting fractions to obtain        salt-exchanged semaglutide.

Suitable conditions for steps (a) to (c) for the second aspect are thesame as ones as defined and described in the first aspect.

B. Purification of Liraglutide

In a third aspect, the present invention provides a process forpurifying liraglutide. The process includes:

-   -   (a) dissolving crude liraglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting purified liraglutide in fractions.

In some embodiments, the halogenated solvent in step (a) is selectedfrom the group consisting of fluorinated acid, fluorinated alcohol,chlorinated solvent, and mixtures thereof.

In some embodiments, the fluorinated alcohol is selected from the groupconsisting of 2,2,2-trifluoroethanol (TFE),1,1,1,3,3,3-hexafluoro-2-propanol (HFIP),2,2,3,3-tetrafluoro-1-propanol, 2,2,3,3,3-pentafluoro-1-propanol,pentafluorophenol, nonafluoro-tert-butyl alcohol,3,3,4,4,4-pentafluoro-1-butanol, 4,4,5,5,5-pentafluoro-1-pentanol,2,2,3,4,4,4-hexafluoro-1-butanol,1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol,1,1,1,3,3,3-hexafluoro-2-phenyl-2-propanol, and mixtures thereof.

In some embodiments, the chlorinated solvent is 2,2,2-trichloroethanol.

In some embodiments, the fluorinated acid is trifluoroacetic acid.

In some embodiments, the solution in step (a) includes 5-35% fluorinatedalcohol and 0.1-1% fluorinated acid by volume. In some embodiments, thesolution in step (a) includes 15-20% 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) and 0.1-1% fluorinated acid by volume. In some embodiments, thesolution in step (a) includes 20-30% 2,2,2-trifluoroethanol (TFE) and0.1-1% fluorinated acid by volume. In some embodiments, the solution instep (a) includes 15-20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and0.1-1% trifluoroacetic acid by volume. In some embodiments, the solutionin step (a) includes 20-30% 2,2,2-trifluoroethanol (TFE) and 0.1-1%trifluoroacetic acid by volume. In some embodiments, the solution instep (a) includes 15-20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and0.1-0.3% trifluoroacetic acid by volume. In some embodiments, thesolution in step (a) includes 20-30% 2,2,2-trifluoroethanol (TFE) and0.1-0.3% trifluoroacetic acid by volume. In some embodiments, thesolution in step (a) includes about 17%1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and about 0.2% trifluoroaceticacid by volume. In some embodiments, the solution in step (a) includesabout 25% 2,2,2-trifluoroethanol (TFE) and about 0.2% trifluoroaceticacid by volume.

In some embodiments, the RP-HPLC purification system of step (b)includes a first RP-HPLC purification and a second RP-HPLC purification;the first RP-HPLC purification is conducted at a pH value of <7; and thesecond RP-HPLC purification is conducted at a pH value of >7.

In some embodiments, the first RP-HPLC purification is conducted at a pHvalue of <7; and the second RP-HPLC purification is conducted at a pHvalue of >about 7. In some embodiments, the first RP-HPLC purificationis conducted at a pH value of <3; and the second RP-HPLC purification isconducted at a pH value of >about 7. In some embodiments, the firstRP-HPLC purification is conducted at a pH value of <3; and the secondRP-HPLC purification is conducted at a pH value of >about 7.5.

In some embodiments, the first RP-HPLC purification uses a mobile phaseA including trifluoroacetic acid and water and a mobile phase Bincluding trifluoroacetic acid and acetonitrile.

In some embodiments, the mobile phase A and mobile phase B include each0.1-1% trifluoroacetic acid by volume. In some embodiments, the mobilephase A and mobile phase B include each 0.1-0.5% trifluoroacetic acid byvolume. In some embodiments, the mobile phase A and mobile phase Binclude each 0.1-0.3% trifluoroacetic acid by volume. In someembodiments, the mobile phase A and mobile phase B include each about0.2% trifluoroacetic acid by volume. In some embodiments, the mobilephase A is a solution of 0.2% trifluoroacetic acid in water; and mobilephase B is a solution of about 0.2% trifluoroacetic acid inacetonitrile.

In some embodiments, the mobile phase B is eluted at a linear gradientof from 5-25% to 80-100% by volume. In some embodiments, the mobilephase B is eluted at a linear gradient of from 5-15% to 90-100% byvolume. In some embodiments, the mobile phase B is eluted at a lineargradient of from 5-25% to 80-100% by volume, wherein the mobile phase Ais a solution of 0.2% trifluoroacetic acid in water; and mobile phase Bis a solution of about 0.2% trifluoroacetic acid in acetonitrile. Insome embodiments, the mobile phase B is eluted at a linear gradient offrom 5-15% to 90-100% by volume, wherein the mobile phase A is asolution of 0.2% trifluoroacetic acid in water; and mobile phase B is asolution of about 0.2% trifluoroacetic acid in acetonitrile. In someembodiments, the mobile phase B is eluted at a linear gradient of fromabout 10% to 95% by volume, wherein the mobile phase A is a solution of0.2% trifluoroacetic acid in water; and mobile phase B is a solution ofabout 0.2% trifluoroacetic acid in acetonitrile. In some embodiments,the mobile phase B is eluted at a linear gradient of from about 10% to95% by volume according to the gradient of Table 3, wherein the mobilephase A is a solution of 0.2% trifluoroacetic acid in water; and mobilephase B is a solution of about 0.2% trifluoroacetic acid inacetonitrile.

In some embodiments, the second RP-HPLC purification uses a mobile phaseC including an ammonium salt or a phosphate salt and a mobile phase Dincluding acetonitrile. In some embodiments, the second RP-HPLCpurification uses a mobile phase C including an ammonium salt and amobile phase D including acetonitrile. In some embodiments, the secondRP-HPLC purification uses a mobile phase C including a phosphate saltand a mobile phase D including acetonitrile. In some embodiments, thesecond RP-HPLC purification uses a mobile phase C and a mobile phase D,each of which includes an ammonium salt or a phosphate salt. In someembodiments, the second RP-HPLC purification uses a mobile phase C and amobile phase D, each of which includes an ammonium salt. In someembodiments, the second RP-HPLC purification uses a mobile phase Cincluding an aqueous solution of an ammonium salt and a mobile phase Dincluding an aqueous solution of an ammonium salt and acetonitrile. Insome embodiments, the second RP-HPLC purification uses a mobile phase Cincluding an aqueous solution of a phosphate salt and a mobile phase Dbeing acetonitrile.

In some embodiments, the mobile phase C further includes acetonitrile inan amount of from 1% to 10% by volume. In some embodiments, the mobilephase C further includes acetonitrile in an amount of from 3% to 7% byvolume. In some embodiments, the mobile phase C further includesacetonitrile in an amount of about 5% by volume.

In some embodiments, the mobile phase D further includes an aqueoussolution of an ammonium salt in an amount of from 10% to 60% by volume.In some embodiments, the mobile phase D further includes an aqueoussolution of an ammonium salt in an amount of from 20% to 60%, 30% to60%, 40% to 60%, or about 50% by volume. In some embodiments, the mobilephase D further includes an aqueous solution of an ammonium salt in anamount of about 50% by volume.

In some embodiments, the ammonium salt is selected from the groupconsisting of ammonium formate, ammonium acetate, and mixtures thereof.In some embodiments, the ammonium salt is ammonium formate.

In some embodiments, the phosphate salt is selected from the groupconsisting of sodium phosphate tribasic, sodium phosphate dibasic,sodium phosphate monobasic, potassium phosphate tribasic, potassiumphosphate dibasic, potassium phosphate monobasic, ammonium phosphatetribasic, ammonium phosphate dibasic, ammonium phosphate monobasic, andmixtures thereof. In some embodiments, the phosphate salt is selectedfrom the group consisting of sodium phosphate dibasic, sodium phosphatemonobasic, potassium phosphate dibasic, potassium phosphate monobasic,ammonium phosphate dibasic, ammonium phosphate monobasic, and mixturesthereof. In some embodiments, the phosphate salt is selected from thegroup consisting of potassium phosphate dibasic, potassium phosphatemonobasic, and mixtures thereof. In some embodiments, the phosphate saltis a mixture of potassium phosphate dibasic and potassium phosphatemonobasic.

In some embodiments, the mobile phase C includes the ammonium salt orphosphate salt in a concentration of from 5 to 50 mM in water. In someembodiments, the mobile phase C includes the ammonium salt or phosphatesalt in a concentration of from 10 to 40 mM in water. In someembodiments, the mobile phase C includes the ammonium salt or phosphatesalt in a concentration of from 10 to 30 mM, from 15 to 25 mM, or about20 mM in water. In some embodiments, the mobile phase C includes theammonium salt or phosphate salt in a concentration of about 20 mM inwater. In some embodiments, the mobile phase C includes the ammoniumsalt in a concentration of about 20 mM in water. In some embodiments,the mobile phase C includes an aqueous solution of 20 mM ammonium salt.In some embodiments, the mobile phase C includes the phosphate salt in aconcentration of about 20 mM in water. In some embodiments, the mobilephase C includes an aqueous solution of 20 mM phosphate salt.

In some embodiments, the mobile phase C has a pH value of from about 7.5to about 9.0 or from about 7.5 to about 8.5. In some embodiments, themobile phase C has a pH value of from about 7.5 to about 8.5. In someembodiments, the mobile phase C has a pH value of about 8.5. In someembodiments, the mobile phase C has a pH value of about 7.9.

In some embodiments, mobile phase C includes an aqueous solution ofammonium formate. In some embodiments, mobile phase C includes anaqueous solution of 20 mM ammonium formate. In some embodiments, mobilephase C includes an aqueous solution of 20 mM ammonium formate; and hasa pH value of about 8.5. In some embodiments, mobile phase C includesacetonitrile and an aqueous solution of 20 mM ammonium formate; and hasa pH value of about 8.5, wherein the aqueous solution and acetonitrilehas a ratio of 95 to 5 by volume.

In some embodiments, mobile phase C includes an aqueous solution ofpotassium phosphate dibasic and potassium phosphate monobasic. In someembodiments, mobile phase C includes an aqueous solution of potassiumphosphate dibasic and potassium phosphate monobasic in a concentrationof 20 mM. In some embodiments, mobile phase C includes an aqueoussolution of potassium phosphate dibasic and potassium phosphatemonobasic in a concentration of 20 mM; and has a pH value of about 7.9.In some embodiments, mobile phase C includes acetonitrile and an aqueoussolution of potassium phosphate dibasic and potassium phosphatemonobasic in a concentration of 20 mM; and has a pH value of about 7.9,wherein potassium phosphate dibasic and potassium phosphate monobasichas a ratio of 12 to 1 by weight, and the aqueous solution andacetonitrile has a ratio of 95 to 5 by volume.

In some embodiments, the mobile phase D has a pH value of from about 7.5to about 9.0 or from about 7.5 to about 8.5. In some embodiments, themobile phase D has a pH value of from about 7.5 to about 8.5. In someembodiments, the mobile phase D has a pH value of about 8.5.

In some embodiments, mobile phase D further includes an aqueous solutionof ammonium formate. In some embodiments, mobile phase D furtherincludes an aqueous solution of 20 mM ammonium formate. In someembodiments, mobile phase D further includes an aqueous solution of 20mM ammonium formate; and has a pH value of about 8.5. In someembodiments, mobile phase D includes an aqueous solution of 20 mMammonium formate and acetonitrile; and has a pH value of about 8.5,wherein the aqueous solution and acetonitrile has a ratio of 50 to 50 byvolume.

In some embodiments, mobile phase D is acetonitrile.

In some embodiments, mobile phase C includes an aqueous solution of 20mM ammonium formate and acetonitrile and has a pH value of about 8.5;and mobile phase D includes an aqueous solution of 20 mM ammoniumformate and acetonitrile and has a pH value of about 8.5, wherein theaqueous solution and acetonitrile in the mobile phase C has a ratio of95 to 5 by volume and the aqueous solution and acetonitrile in themobile phase D has a ratio of 50 to 50 by volume.

In some embodiments, mobile phase C includes acetonitrile and an aqueoussolution of potassium phosphate dibasic and potassium phosphatemonobasic in a concentration of 20 mM and has a pH value of about 7.9;and mobile phase D is acetonitrile, wherein potassium phosphate dibasicand potassium phosphate monobasic has a ratio of 12 to 1 by weight, andthe aqueous solution and acetonitrile in the mobile phase C has a ratioof 95 to 5 by volume.

In some embodiments, the mobile phase D is eluted at linear gradientfrom 0-10% to 50-100% by volume. In some embodiments, the mobile phase Dis eluted at linear gradient from 0% to 100% by volume, wherein mobilephase C includes an aqueous solution of 20 mM ammonium formate andacetonitrile and has a pH value of about 8.5; and mobile phase Dincludes an aqueous solution of 20 mM ammonium formate and acetonitrileand has a pH value of about 8.5, wherein the aqueous solution andacetonitrile in the mobile phase C has a ratio of 95 to 5 by volume andthe aqueous solution and acetonitrile in the mobile phase D has a ratioof 50 to 50 by volume. In some embodiments, the mobile phase D is elutedat linear gradient from 0% to 100% by volume according to the gradientof Table 4, wherein mobile phase C includes an aqueous solution of 20 mMammonium formate and acetonitrile and has a pH value of about 8.5; andmobile phase D includes an aqueous solution of 20 mM ammonium formateand acetonitrile and has a pH value of about 8.5, wherein the aqueoussolution and acetonitrile in the mobile phase C has a ratio of 95 to 5by volume and the aqueous solution and acetonitrile in the mobile phaseD has a ratio of 50 to 50 by volume. In some embodiments, the mobilephase D is eluted at linear gradient from 5% to 70% by volume, whereinmobile phase C includes acetonitrile and an aqueous solution ofpotassium phosphate dibasic and potassium phosphate monobasic in aconcentration of 20 mM and has a pH value of about 7.9; and mobile phaseD is acetonitrile, wherein potassium phosphate dibasic and potassiumphosphate monobasic has a ratio of 12 to 1 by weight, and the aqueoussolution and acetonitrile in the mobile phase C has a ratio of 95 to 5by volume. In some embodiments, the mobile phase D is eluted at lineargradient from 5% to 70% by volume according to the gradient Table 5,wherein mobile phase C includes acetonitrile and an aqueous solution ofpotassium phosphate dibasic and potassium phosphate monobasic in aconcentration of 20 mM and has a pH value of about 7.9; and mobile phaseD is acetonitrile, wherein potassium phosphate dibasic and potassiumphosphate monobasic has a ratio of 12 to 1 by weight, and the aqueoussolution and acetonitrile in the mobile phase C has a ratio of 95 to 5by volume.

In some embodiments, the purified liraglutide in fractions has a pHvalue of from about 7.5 to about 8.5. In some embodiments, the purifiedliraglutide in fractions has a pH value of from about 8.0 to about 8.5.In some embodiments, the purified liraglutide in fractions has a pHvalue of from about 7.5 to about 8.0.

The flow rate of the first and second purification depends on the sizeof the RP-HPLC preparation column. In some embodiments, the flow rate ofthe first purification is from about 50 to 200 mL/min, when the columnhas a diameter and length of from 5 cm×22 cm to 8 cm×12.6 cm. In someembodiments, the flow rate of the first purification is from about 100to 160 mL/min, when the column has a diameter and length of from 5 cm×22cm to 8 cm×12.6 cm. In some embodiments, the flow rate of the firstpurification is about 104 mL/min, when the column has a diameter andlength of 5 cm×22 cm. In some embodiments, the flow rate of the firstpurification is about 152 mL/min, when the column has a diameter andlength of 8 cm×12.6 cm. In some embodiments, the flow rate of the firstpurification is from 4 to 8 mL/min, when the column has a diameter andlength of 10 mm×250 mm. In some embodiments, the flow rate of the firstpurification is about 4.7 mL/min, when the column has a diameter andlength of 10 mm×250 mm. In some embodiments, the flow rate of the secondpurification is from about 50 to 200 mL/min, when the column has adiameter and length of from 5 cm×22 cm to 8 cm×12.6 cm. In someembodiments, the flow rate of the second purification is from about 100to 160 mL/min, when the column has a diameter and length of from 5 cm×22cm to 8 cm×12.6 cm. In some embodiments, the flow rate of the secondpurification is about 152 mL/min, when the column has a diameter andlength of 8 cm×12.6 cm. In some embodiments, the flow rate of the secondpurification is from about 0.3 to 0.5 mL/min, when the column has adiameter and length of 4.6 mm×100 mm. In some embodiments, the flow rateof the second purification is about 0.4 mL/min, when the column has adiameter and length of 4.6 mm×100 mm.

In some embodiments, the crude liraglutide of step (a) is obtained fromsolid-phase synthesis.

In some embodiments, further comprising:

-   -   (c) subjecting the fractions in step (b) to a third RP-HPLC        purification using a mobile phase E comprising a sodium salt,        water, and acetonitrile; and collecting fractions to obtain        salt-exchanged liraglutide.

In some embodiments, the sodium salt is sodium phosphate dibasic.

In some embodiments, the mobile phase E includes an aqueous solution ofsodium phosphate dibasic. In some embodiments, mobile phase E includesan aqueous solution of sodium phosphate dibasic in a concentration of 20mM. In some embodiments, mobile phase E includes an aqueous solution ofsodium phosphate dibasic in a concentration of 20 mM; and has a pH valueof about 8.0. In some embodiments, mobile phase E (as E1) includesacetonitrile and an aqueous solution of sodium phosphate dibasic in aconcentration of 20 mM; and has a pH value of about 8.0, wherein theaqueous solution and acetonitrile has a ratio of 95 to 5 by volume. Insome embodiments, mobile phase E (as E2) includes acetonitrile and anaqueous solution of sodium phosphate dibasic in a concentration of 20mM; and has a pH value of about 8.0, wherein the aqueous solution andacetonitrile has a ratio of 50 to 50 by volume.

In some embodiments, step (c) is conducted by applying the mobile phaseE1 followed by the mobile phase E2, thereby providing a salt-exchangedliraglutide in fractions.

The flow rate of the third purification depends on the size of theRP-HPLC preparation column. In some embodiments, the flow rate of thethird purification is from 4 to 8 mL/min, when the column has a diameterand length of 10 mm×250 mm. In some embodiments, the flow rate of thefirst purification is about 4.7 mL/min, when the column has a diameterand length of 10 mm×25 cm.

In some embodiments, the salt-exchanged liraglutide in fractions has apH value of from about 7.5 to about 8.0.

In some embodiments, the first, second, and third RP-HPLC purificationshave the same stationary phase.

In a fourth aspect, the present application provides a process forpurifying liraglutide. The process includes:

-   -   (a) dissolving crude liraglutide in a solution comprising a        halogenated solvent; and    -   (b) subjecting the solution to a RP-HPLC purification system,        and collecting the purified liraglutide fractions, wherein the        RP-HPLC purification system includes a first RP-HPLC        purification and a second RP-HPLC purification; and    -   (c) subjecting the fractions in step (b) to a third RP-HPLC        purification using a mobile phase E including a sodium salt,        water, and acetonitrile; and collecting fractions to obtain        salt-exchanged liraglutide.

Suitable conditions for steps a) to (c) for the fourth aspect are thesame as ones as defined and described in the third aspect.

IV. Embodiments

Embodiment 21: A process for purifying liraglutide, comprising:

-   (a) dissolving crude liraglutide in a solution comprising a    halogenated solvent; and-   (b) subjecting the solution to a RP-HPLC purification system, and    collecting purified liraglutide in fractions.

Embodiment 22: The process according to Embodiment 21, wherein thehalogenated solvent in step (a) is selected from the group consisting ofa fluorinated acid, a fluorinated alcohol, a chlorinated solvent, andmixtures thereof.

Embodiment 23: The process according to Embodiment 22, wherein thefluorinated alcohol is selected from the group consisting of2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP),2,2,3,3-tetrafluoro-1-propanol, 2,2,3,3,3-pentafluoro-1-propanol,pentafluorophenol, nonafluoro-tert-butyl alcohol,3,3,4,4,4-pentafluoro-1-butanol, 4,4,5,5,5-pentafluoro-1-pentanol,2,2,3,4,4,4-hexafluoro-1-butanol,1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol,1,1,1,3,3,3-hexafluoro-2-phenyl-2-propanol, and mixtures thereof.

Embodiment 24: The process according to Embodiment 22, wherein thechlorinated solvent is 2,2,2-trichloroethanol.

Embodiment 25: The process according to Embodiment 22, wherein thefluorinated acid is trifluoroacetic acid.

Embodiment 26: The process according to Embodiment 22, wherein thesolution in step (a) comprises 5-35% fluorinated alcohol and 0.1-1%fluorinated acid by volume.

Embodiment 27: The process according to Embodiment 26, wherein thesolution in step (a) comprises 15-20% 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) and 0.1-1% fluorinated acid by volume.

Embodiment 28: The process according to Embodiment 26, wherein thesolution in step (a) comprises 20-30% 2,2,2-trifluoroethanol (TFE) and0.1-1% fluorinated acid by volume.

Embodiment 29: The process according to Embodiment 21, wherein theRP-HPLC purification system of step (b) comprises a first RP-HPLCpurification and a second RP-HPLC purification; the first RP-HPLCpurification is conducted at a pH value of <7; and the second RP-HPLCpurification is conducted at a pH value of >7.

Embodiment 30: The process according to Embodiment 29, wherein the firstRP-HPLC purification is conducted at a pH value of <3; and the secondRP-HPLC purification is conducted at a pH value of >about 7.5.

Embodiment 31: The process according to Embodiment 29, wherein the firstRP-HPLC purification uses a mobile phase A comprising trifluoroaceticacid and water and a mobile phase B comprising trifluoroacetic acid andacetonitrile.

Embodiment 32: The process according to Embodiment 31, wherein themobile phase A and mobile phase B comprise each 0.1-1% trifluoroaceticacid by volume.

Embodiment 33: The process according to Embodiment 32, wherein themobile phase B is eluted at a linear gradient of from 5-25% to 80-100%by volume.

Embodiment 34: The process according to Embodiment 29, wherein thesecond RP-HPLC purification uses a mobile phase C comprising an ammoniumsalt or a phosphate salt and a mobile phase D comprising acetonitrile.

Embodiment 35: The process according to Embodiment 34, wherein theammonium salt is selected from the group consisting of ammonium formate,ammonium acetate, and mixtures thereof.

Embodiment 36: The process according to Embodiment 34, wherein thephosphate salt is selected from the group consisting of sodium phosphatetribasic, sodium phosphate dibasic, sodium phosphate monobasic,potassium phosphate tribasic, potassium phosphate dibasic, potassiumphosphate monobasic, ammonium phosphate tribasic, ammonium phosphatedibasic, ammonium phosphate monobasic, and mixtures thereof.

Embodiment 37: The process according to Embodiment 34, wherein themobile phase C comprises the ammonium salt or phosphate salt in aconcentration of from 5 to 50 mM in water.

Embodiment 38: The process according to Embodiment 34, wherein themobile phase C has a pH value of from about 7.5 to about 9.0 or fromabout 7.5 to about 8.5.

Embodiment 39: The process according to Embodiment 34, wherein themobile phase D is eluted at linear gradient from 0-10% to 50-100% byvolume.

Embodiment 40: The process according to Embodiment 34, wherein thepurified liraglutide in fractions has a pH value of from about 7.5 toabout 8.5.

Embodiment 41: The process according to Embodiment 21, furthercomprising:

-   (c) subjecting the fractions in step (b) to a third RP-HPLC    purification using a mobile phase E comprising a sodium salt, water,    and acetonitrile; and collecting fractions to obtain salt-exchanged    liraglutide.

Embodiment 42: The process according to Embodiment 41, wherein thesodium salt is sodium phosphate dibasic.

Embodiment 43: The process according to Embodiment 41, wherein thesalt-exchanged liraglutide in fractions has a pH value of from about 7.5to about 8.0.

V. EXAMPLES

The following examples are provided to further illustrate, but not tolimit this invention.

Example 1

Sample Pretreatment:

The crude semaglutide was obtained from solid-phase synthesis with apurity of 36.61%. 189.0 mg crude semaglutide was dissolved in 3.15 mLtriflouroethanol (TFE) solution (25% TFE in 0.2% trifluoroacetic acidsolution). The mixture was then stirred at room temperature (RT) forabout 2 hrs.

First Purification:

The obtained solution was subjected to a RP-HPLC system with apreparative column packed with C8, 10 μm silica having a diameter andlength of 4.6 mm×250 mm. The loading amount was 189.0 mg.

A purification cycle was performed with condition of:

-   -   (1) mobile phase A: 0.2% trifluoroacetic acid (TFA) in water, pH        value of 1-2;    -   (2) mobile phase B: 0.2% TFA in acetonitrile (ACN);    -   (3) flow rate: 1 mL/min;    -   (4) detector: UV at 250 nm and 300 nm; and    -   (5) elution gradient Table 1:

Time Composition (minute:second) MPA (%) by volume MPB (%) by volume 00:00 90 10  10:00 90 10  35:00 65 35  75:00 50 50  75:06 5 95 100:00 595

Fractions containing semaglutide with 85.87% purity were obtained andtransferred to second purification.

Second Purification:

Fractions containing semaglutide with 85.87% purity were adjusted with33% ammonium hydroxide to pH value of 8.3 and diluted with 15 mMammonium formate with pH value of 8.3, and then subjected to a RP-HPLCsystem with a column packed with C8, 10 μm silica having a diameter andlength of 4.6 mm×100 mm. The loading amount was 16.6 mg.

A purification cycle was performed with condition of:

-   -   (1) mobile phase C: 15 mM ammonium formate (AmF) adjusted to pH        value of 8.3;    -   (2) mobile phase D: ACN;    -   (3) flow rate: 0.4 mL/min;    -   (4) detector: UV at 250 nm and 300 nm; and    -   (5) elution gradient Table 2:

Time Composition (minute:second) MPC (%) by volume MPD (%) by volume 00:00 95 5  05:00 95 5  15:00 70 30  65:00 60 40  65:06 30 70 105:00 3070

Fractions containing semaglutide with 95.58% purity and pH value about8.0-8.5 were obtained.

Example 2

Sample Pretreatment:

The crude semaglutide was obtained from solid-phase synthesis with apurity of 30.53%. 187.8 mg crude semaglutide was dissolved in 2.125 mLhexafluoroisopropanol (HFIP) solution (17% HFIP in 0.2% TFA solution).The mixture was then stirred at room temperature (RT) for about 2 hrs.

First Purification:

The obtained solution was subjected to first purification that have thesame purifying condition and method with the Example 1. The loadingamount was 187.8 mg.

Fractions containing semaglutide with 76.43% purity were obtained andtransferred to second purification. The loading amount was 15.8 mg.

Second Purification:

Fractions containing semaglutide with 76.43% purity were then subjectedto second purification that have the same purifying condition and methodwith the Example 1. Fractions containing semaglutide with 94.74% purityand pH value about 8.0-8.5 were obtained.

Example 3

Sample Pretreatment:

The crude liraglutide was obtained from solid-phase synthesis with apurity of 44.6%. 30 g crude liraglutide was dissolved in 500 mLtriflouroethanol (TFE) solution (25% TFE in 0.2% trifluoroacetic acid).The mixture was then stirred at room temperature (RT) for about 2 hrs.

First Purification:

The obtained solution was loaded onto a RP-HPLC preparative columnpacked with C8, 10 μm silica having a diameter and length of 8 cm×12.6cm. The loading amount was 27.1 g.

A purification cycle was performed with condition of:

-   -   (1) mobile phase A: 0.2% trifluoroacetic acid (TFA) in water;    -   (2) mobile phase B: 0.2% TFA in acetonitrile (ACN);    -   (3) flow rate: 152 mL/min; and    -   (4) elution gradient Table 3:

Time Composition (minute:second) MPA (%) by volume MPB (%) by volume 00:00 90 10  10:00 90 10  40:00 60 40  80:00 45 55  80:06 5 95 100:06 595

Fractions containing liraglutide with 90.58% purity were obtained andtransferred to second purification.

Second Purification:

Fractions containing liraglutide with 90.58% purity were adjusted with33% Ammonium to pH value of 8.5 and diluted with 20 mM ammonium formatewith pH value of 8.5, and then loaded onto RP-HPLC column packed withC8, 10 μm silica having a diameter and length of 8 cm×12.6 cm. Theloading amount was 6.2 g.

A purification cycle was performed with condition of:

-   -   (1) mobile phase C: 20 mM ammonium formate (AmF) adjusted to pH        value of 8.5 and diluted with ACN to the ratio of 95/5 (V/V);    -   (2) mobile phase D: 20 mM AmF adjusted to pH value of 8.5 and        diluted with ACN to the ratio of 50/50 (V/V);    -   (3) flow rate: 152 mL/min; and    -   (4) elution gradient Table 4:

Time Composition (minute:second) MPC (%) by volume MPD (%) by volume 00:00 100 0  05:00 100 0  15:00 45 55  65:00 23 77  75:00 0 100 120:000 100

Fractions containing liraglutide with 98.48% purity and pH value about8.0-8.5 were obtained and transferred to third purification (saltexchange).

Third Purification (Salt Exchange):

Fractions containing liraglutide with 98.48% purity were loaded ontoRP-HPLC column packed with C8, 10 μm silica having a diameter and lengthof 10 mm×250 mm with a flow rate of 4.7 mL/min. The loading amount was1.1 g. After loaded, the column was flushed with 20 mM sodium phosphatedibasic solution (pH=8.0) comprising 5% acetonitrile, and then elutedwith 20 mM sodium phosphate dibasic solution (pH=8.0) comprising 50%acetonitrile. Thus, salt-exchanged liraglutide with pH value about7.5-8.0 was obtained, and then lyophilized, resulting in 3.3 gliraglutide concentrate with 98.48% purity.

Example 4

Sample Pretreatment:

The crude liraglutide was obtained from solid-phase synthesis with apurity of 33.8%. 19.5 gram crude liraglutide was dissolved in 238 mLhexafluoroisopropanol (HFIP) solution (17% HFIP in 0.2% TFA solution).The mixture was then stirred at room temperature (RT) for about 2 hrs.

First Purification:

The obtained solution was loaded onto a RP-HPLC preparative columnpacked with C8, 10 μm silica having a diameter and length of 5 cm×22 cm.The loading amount was 19.5 g.

A purification cycle was performed with condition of:

-   -   (1) mobile phase A: 0.2% trifluoroacetic acid (TFA) in water;    -   (2) mobile phase B: 0.2% TFA in acetonitrile (ACN);    -   (3) flow rate: 104 mL/min; and    -   (4) elution gradient Table 3:

Time Composition (minute:second) MPA (%) by volume MPB (%) by volume 00:00 90 10  10:00 90 10  40:00 60 40  80:00 45 55  80:06 5 95 100:06 595

Fractions containing liraglutide with 89.65% purity were obtained andtransferred to second purification. The loading amount was 4.3 g.

Second Purification and Third Purification (Salt Exchange):

Fractions containing liraglutide with 89.65% purity were then subjectedto second purification and third purification (salt exchange) that havethe same purifying condition and method with the Example 3. Thus,salt-exchanged liraglutide with pH value about 7.5-8.0 was obtained, andthen lyophilized, resulting in 3.7 g liraglutide concentrate with 97.23%purity.

Example 5

Sample Pretreatment:

The crude liraglutide was obtained from solid-phase synthesis with apurity of 33.8%. 990.3 milligram crude liraglutide was dissolved in11.22 mL hexafluoroisopropanol (HFIP) solution (17% HFIP in 0.2% TFAsolution). The mixture was then stirred at room temperature (RT) forabout 2 hrs.

First Purification:

The obtained solution was loaded onto a RP-HPLC preparative columnpacked with C8, 10 μm silica having a diameter and length of 10 mm×25cm. The loading amount was 0.99 g.

A purification cycle was performed with condition of:

-   -   (1) mobile phase A: 0.2% trifluoroacetic acid (TFA) in water;    -   (2) mobile phase B: 0.2% TFA in acetonitrile (ACN);    -   (3) flow rate: 4.7 mL/min; and    -   (4) elution gradient Table 3:

Time Composition (minute:second) MPA (%) by volume MPB (%) by volume 00:00 90 10  10:00 90 10  40:00 60 40  80:00 45 55  80:06 5 95 100:06 595

Fractions containing liraglutide with 92.17% purity were obtained andtransferred to second purification. The loading amount was 41.1milligram.

Second Purification:

Fractions containing liraglutide with 92.17% purity were diluted withmobile phase C, and then loaded onto RP-HPLC column packed with C8, 10μm silica having a diameter and length of 4.6 mm×100 mm.

A purification cycle was performed with condition of:

-   -   (1) mobile phase C: 20 mM potassium phosphate dibasic        (K₂HPO₄)/potassium phosphate monobasic (KH₂PO₄)=12/1, adjusted        to pH value of 7.9, and diluted with ACN to the ratio of 95/5        (V/V);    -   (2) mobile phase D: ACN;    -   (3) flow rate: 0.4 mL/min; and    -   (3) elution gradient Table 5:

Time Composition (minute:second) MPC (%) by volume MPD (%) by volume 00:00 95 5  10:00 95 5  23:00 80 20  98:00 55 45  98:06 30 70 118:06 3070

Fractions containing liraglutide with 97% purity and pH value about7.5-8.0 were obtained and transferred to third purification (saltexchange).

Third Purification (Salt Exchange):

Third purification (salt exchange) has the same purifying condition andmethod with the Example 3. Thus, salt-exchanged liraglutide with pHvalue about 7.5-8.0 was obtained, and then lyophilized.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A process for purifying semaglutide, comprising:(a) dissolving crude semaglutide in a solution comprising a halogenatedsolvent; and (b) subjecting the solution to a RP-HPLC purificationsystem, and collecting purified semaglutide in fractions.
 2. The processaccording to claim 1, wherein the halogenated solvent in step (a) isselected from the group consisting of a fluorinated acid, a fluorinatedalcohol, a chlorinated solvent, and mixtures thereof.
 3. The processaccording to claim 2, wherein the fluorinated alcohol is selected fromthe group consisting of 2,2,2-trifluoroethanol (TFE),1,1,1,3,3,3-hexafluoro-2-propanol (HFIP),2,2,3,3-tetrafluoro-1-propanol, 2,2,3,3,3-pentafluoro-1-propanol,pentafluorophenol, nonafluoro-tert-butyl alcohol,3,3,4,4,4-pentafluoro-1-butanol, 4,4,5,5,5-pentafluoro-1-pentanol,2,2,3,4,4,4-hexafluoro-1-butanol,1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol,1,1,1,3,3,3-hexafluoro-2-phenyl-2-propanol, and mixtures thereof.
 4. Theprocess according to claim 2, wherein the chlorinated solvent is2,2,2-trichloroethanol.
 5. The process according to claim 2, wherein thefluorinated acid is trifluoroacetic acid.
 6. The process according toclaim 2, wherein the solution in step (a) comprises 5-35% fluorinatedalcohol and 0.1-1% fluorinated acid by volume.
 7. The process accordingto claim 6, wherein the solution in step (a) comprises 15-20%1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 0.1-1% fluorinated acid byvolume.
 8. The process according to claim 6, wherein the solution instep (a) comprises 20-30% 2,2,2-trifluoroethanol (TFE) and 0.1-1%fluorinated acid by volume.
 9. The process according to claim 1, whereinthe RP-HPLC purification system of step (b) comprises a first RP-HPLCpurification and a second RP-HPLC purification; the first RP-HPLCpurification is conducted at a pH value of <7; and the second RP-HPLCpurification is conducted at a pH value of >7.
 10. The process accordingto claim 9, wherein the first RP-HPLC purification is conducted at a pHvalue of <3; and the second RP-HPLC purification is conducted at a pHvalue of >about 7.5.
 11. The process according to claim 9, wherein thefirst RP-HPLC purification uses a mobile phase A comprisingtrifluoroacetic acid and water and a mobile phase B comprisingtrifluoroacetic acid and acetonitrile.
 12. The process according toclaim 11, wherein the mobile phase A and mobile phase B comprise each0.1-1% trifluoroacetic acid by volume.
 13. The process according toclaim 12, wherein the mobile phase B is eluted at a linear gradient offrom 5-25% to 80-100% by volume.
 14. The process according to claim 9,wherein the second RP-HPLC purification uses a mobile phase C comprisingan ammonium salt or a phosphate salt and a mobile phase D comprisingacetonitrile.
 15. The process according to claim 14, wherein theammonium salt is selected from the group consisting of ammonium formate,ammonium acetate, and mixtures thereof.
 16. The process according toclaim 14, wherein the phosphate salt is selected from the groupconsisting of sodium phosphate tribasic, sodium phosphate dibasic,sodium phosphate monobasic, potassium phosphate tribasic, potassiumphosphate dibasic, potassium phosphate monobasic, ammonium phosphatetribasic, ammonium phosphate dibasic, ammonium phosphate monobasic, andmixtures thereof.
 17. The process according to claim 14, wherein themobile phase C comprises the ammonium salt or phosphate salt in aconcentration of from 5 to 50 mM in water.
 18. The process according toclaim 14, wherein the mobile phase C has a pH value of from about 7.5 toabout 9.0 or from about 7.5 to about 8.5.
 19. The process according toclaim 14, wherein the mobile phase D is eluted at linear gradient from0-10% to 50-100% by volume.
 20. The process according to claim 14,wherein the purified semaglutide in fractions has a pH value of fromabout 7.5 to about 8.5.